Pairs of nucleic acid probes with interactive signaling moieties and nucleic acid probes with enhanced hybridization efficiency and specificity

A method of detecting a sequence of interest in a sample, said method comprising contacting the sample with an acceptor probe and a donor probe,

• wherein the acceptor and donor probes comprise polynucleotides that are hybridizable to non-overlapping portions of a target polynucleotide, wherein the donor probe comprises a sequence hybridizable to the sequence of interest,
• wherein said acceptor probe comprises an acceptor moiety and said donor probe comprises a donor moiety, and wherein hybridization of both the donor and acceptor probes to a target polynucleotide comprising the sequence of interest allows the donor moiety and the acceptor moiety to interact mode, wherein the interaction is detectable, and
• wherein a mismatch between the sequence of interest and the donor probe substantially prevents a portion of the donor probe from hybridizing to the sequence of interest such that the interaction of the donor moiety and the acceptor moiety is diminished, and wherein said portion of the donor probe is the portion that spans the sequence of the target polynucleotide between the mismatch site and hybridization site of the opposing end of the acceptor probe.

A nucleic acid probe comprising a nucleic acid with first and second ends, a spacer with first and second ends, and a minor groove binder (MGB), wherein the first end of the spacer is linked to the nucleic acid at or near the first end of the nucleic acid, the second end of the spacer is linked to a solid substrate, and the MGB is linked to the nucleic acid at or near the second end of the nucleic acid.